Most neuronal systems are innervated by multiple afferent pathways that utilize a wide variety of neurotransmitters and neuropeptides. In addition, other agents such as hormones and cytokines influence neurons via non-synaptic mechanisms, and many neurons release multiple neuroactive agents. A major challenge for neuroscientists is to understand the mechanisms that allow target neurons to integrate these regulatory signals into appropriate responses. This proposal focuses on two aspects of this using the vasopressin (VP) neurons of the hypothalamo-neurohypophseal system (HNS) as a model system: 1) The interactions between ionotropic and G-protein mediated afferents; and 2) the modulatory effects of steroid hormones on these afferent signals. The proposed experiments build on two important findings from the past funding cycle: 1) Impressive synergism between ATP and phenylephrine (to mimic the action of norepinephrine on alpha1-adrenergic receptors) and preliminary evidence for synergism between glutamate and AII; and 2) Evidence that estrogen receptor beta (ER-beta) is inhibitory to VP release and that its expression is inversely correlated with osmotic stimulation of the HNS. The specific aims of the proposal are: 1. To address the hypothesis that the synergistic responses between ATP plus PE and glutamate plus AII reflect activation of similar intracellular signal cascades that converge to increase intracellular Ca++. 2. To evaluate the hypothesis that the synergistic responses to ATP plus PE and glutamate plus AII reflect alterations in intracellular Ca++. 3. To test the hypothesis that gonadal steroids inhibit the response to ionotropic agents that allow Ca++ influx via an ER-beta mediated mechanism. 4. To test the hypothesis that ER-beta expression is inhibited by activation of afferent pathways that stimulate VP and/or OT release. Perifused explants of the HNS will be used to study VP release. Live cell imaging of SON neurons in HNS explants and acutely dissociated preparations will address the role of Ca++ as an integrator of signal cascades for ionotropic and G-protein coupled ligands. Immunocytochemistry and in situ hybridization techniques will be used to study regulation of ER-beta expression.